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First published online 27 November 2002
doi: 10.1242/jcs.00217

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Furrow microtubules and localized exocytosis in cleaving Xenopus laevis embryos

Department Biological Structure and Function SD, Oregon Health Sciences University, Portland, OR 97201-3097 USA

View larger version (37K): [in a new window]   Fig. 1. Development of new basolateral domain is contemporaneous with cleavage furrowing. Embryo manually stripped of its vitelline envelope was placed on an agarose-coated surface, incubated briefly with FITC-soybean agglutinin and observed via full-spectrum (upper panels) or epifluorescence (lower panels) illumination. The new membrane domain develops as a broad, unpigmented surface along the cleavage plane (A-D). Lectin bound to the surface of devitellinated egg undergoes local concentration at onset of furrowing (bright stripe, E), followed by appearance and expansion on either side of the new, unlabelled domains of the basolateral membrane (dark areas; arrows). (H) The membrane expansion process repeats during second cleavage (arrows). Note that the new/old membrane boundary corresponds closely to the boundary between pigmented and unpigmented regions. Frames approximately 8 minutes apart.

View larger version (148K): [in a new window]   Fig. 4. SEMs of the furrow region. (A,C) Low magnification views of cleaving embryos, with a new membrane domain appearing in the furrow region. Areas indicated by boxes are shown at higher magnification in lower panels. Bars, 100 µm and 50 µm, respectively. (B,D) Region near the center of the furrow. Note the numerous pits (arrows) with raised edges appearing in clusters (asterisks) near the furrow base. Small white structures at the base of the furrow are microvilli. Bars, 10 µm.

View larger version (45K): [in a new window]   Fig. 2. Furrow microtubule bundles directly underlie the growing domain of the new membrane. (A) Furrow ceases ingression in an embryo treated with 0.1 µM latrunculin B. New membrane deposition continues, however, producing broad white stripe. (B) Same embryo, fixed and stained with antitubulin antibody. Montage is series of confocal micrographs running along cleavage furrow from animal pole to horizon. The curvature of the embryo is not apparent in montage because individual frames are single projections of image stacks, each representing several tens of microns of specimen depth. Bar, 50 µm.

View larger version (52K): [in a new window]   Fig. 3. Membrane expansion occurs from a site near the furrow base. A suspension of carbon particles was pipetted over the surface of devitellinated cleaving embryo. A video time-lapse sequence recorded particle movement during new membrane expansion. (A-D) Frames from the sequence, captured at 125 second intervals. (E) Kymograph made by vertically reprojecting the region of the timelapse image stack indicated by dotted box in frame A. Small arrows (a-d) indicate times in the sequence corresponding to panels A-D, respectively. Based on the parallel trajectories indicated, particles near each other travel at nearly identical speeds. Because they do not drift apart, evidently little new membrane insertion occurs between them. Vertical scale bar, 250 µm. Horizontal scale bar, 5 minutes.

View larger version (18K): [in a new window]   Fig. 5. Distribution of exocytotic fusion pores in the new membrane. Values refer to the number of pits counted in successive 10 µm x 50 µm regions of interest relative to the furrow base, without (closed symbols) or with (open symbols) exposure to nocodazole. The embryo was fixed 15 minutes after the beginning of the first cleavage.

View larger version (178K): [in a new window]   Fig. 6. Confocal image of live embryo at the beginning of the second cleavage, continuously bathed in FM1-43 and cytochalasin B. Figure shown is a flat projection of a stack of images captured through 300 µm of specimen depth. Fluorescing FM1-43 accumulates in the new membrane domain of the first cleavage plane (broad vertical stripe), but not in the original membrane domains (asterisk). Localized bright-staining puncta along the new/old membrane boundaries correspond to collections of long microvilli, and do not represent sites of endocytosis (data not shown). Membrane newly inserted along the second cleavage furrow (surrounding arrow) is momentarily less well-labeled than that previously inserted along the first furrow.

View larger version (162K): [in a new window]   Fig. 7. Exocytosis contributes a patch of unlabeled surface to the new plasma membrane domain of an FM1-43-labeled embryo. Three representative sequences, A1-4, B1-4 and C1-4, taken from time-lapse Movie 2 (see http://jcs.biologists.org/supplemental), display a particularly active site recorded near the arrow in Fig. 6. Expanding dark regions (arrows) indicate sites of displacement of FM1-43-labeled membrane. Frames were recorded at 1.8 second intervals. Bar, 10 µm.

View larger version (148K): [in a new window]   Fig. 8. VSVG-containing vesicles collect and undergo exocytosis near furrow bases. Unfertilized eggs were allowed several hours to express injected VSVG mRNA, and then were fertilized and fixed part way through the fourth cleavage. Exocytosis of labeled vesicles was exclusively along nascent basolateral surfaces lining the blastocoel. Vesicle size ranges up to 2.5 µm. Bar, 10 µm.

View larger version (177K): [in a new window]   Fig. 9. D2O-induced membrane expansion. (A) Embryos at 75 minutes post fertilization, prior to application of D2O. Asterisks indicate embryos lacking vitelline envelopes. (B) Following 10 minutes incubation in 60% D2O, devitellinated embryos increase their surface area and spread across the substrate. (C) Side view, obtained by recording down through a 45° prism in a culture dish. Frames are 3 minutes apart.

View larger version (184K): [in a new window]   Fig. 10. Scanning EM of D2O-treated embryo fixed during surface expansion. Low magnification (A) shows that the entire surface retains short microvilli that normally cover the embryo surface. The boxed region is shown at higher magnification in lower panel. Bar, 100 mm. Higher magnification (B) reveals random arrangement of fusion pores between collections of microvilli. Bar, 10 µm.

View larger version (14K): [in a new window]   Fig. 11. D2O-induced membrane expansion requires microtubules. Overall surface areas were calculated from measurements of horizontal profiles of embryos incubated in D2O alone or D2O with 10 µM nocodazole, as described in the text. Values shown are means for three embryos. The black arrow denotes onset of first cleavage in untreated sibling embryos.

View larger version (137K): [in a new window]   Fig. 12. Effect of D2O and nocodazole on microtubule density near the embryo surface. Wholemount confocal microscopy of embryos stained for ß-tubulin. (A) Furrow region of untreated cleaving embryo. (B) The same embryo, non-furrowing surface of animal hemisphere. Inset is vertical resection at the site indicated by the white horizontal line. (C) Animal surface of D2O-treated embryo, showing through-surface view of a microtubule monaster. The inset is a vertical resection that indicates monastral microtubules are within 1 or 2 µm of the surface. (D) Nocodazole treatment completely eliminates surface microtubules. (E) Nocodazole treatment eliminates surface microtubules in a D2O-incubated embryo. Bar, 10 µm.

View larger version (55K): [in a new window]   Fig. 13. Microtubules and -tubulin in a D2O-treated embryo, viewed via wholemount confocal microscopy. (A) ß-tubulin staining. (B) -tubulin staining. (C) Merged frames; red channel, ß-tubulin staining; green, -tubulin staining. Bar, 10 µm.